The importance of cyclic dinucleotides as bacterial second messengers is well established, with cyclic di-GMP (c-di-GMP) now acknowledged as a universal bacterial second messenger. This versatile molecule has been shown to play key roles in cell cycle and differentiation, motility and virulence, as well as in the regulation of biofilm formation and dispersion. Advances in our understanding of c-di-GMP has emerged with the identification, structural characterization, and mechanistic understanding of the catalytic activities of the bacterial enzymes responsible for the synthesis and degradation of this second messenger. Crystal structures of c-di-GMP in the free state and when bound to enzymes responsible for its synthesis and degradation have shown that this second messenger can adopt either monomeric or a dimeric bis-intercalated folds. It appears that formation of c-(3′,5′)-di-GMP from two molecules of GTP occurs via a two-step reaction and formation of 3′,5′-phosphodiester linkages, with two molecules of pyrophosphate as byproducts of the cyclization reaction. Moreover, multiple receptors targeted by c-(3′,5′)-di-GMP and the diverse ways bacteria signal through this second messenger have been identified. Indeed, the field of c-di-GMP study as a second messenger has grown immensely and yielded major advances in our understanding of the physiology and mechanisms of bacterial cyclic dinucleotide signaling over the last two and a half decades. In parallel studies, c-(3′,5′)-di-GMP-specific riboswitches have also been identified, including ones that are involved in cyclic dinucleotide-induced RNA splicing.
There is much interest currently towards gaining a molecular and functional understanding of innate immunity sensors of higher metazoans that recognize nucleic acids in the cytoplasm and trigger type I interferon induction. Cytoplasmic dsDNA of pathogenic bacterial or viral origin, and perhaps also displaced nuclear or mitochondrial DNA following cellular stress, represent such a trigger. These events involving self-nucleic acid recognition in turn could trigger autoimmune diseases such as systemic lupus erythematosus and Sjögren syndrome. Indeed, in recent years many cytoplasmic DNA sensors have been identified, including DAI (DNA-dependent activator of IFN-regulatory factor), LRRFIP1 (leucine-rich repeat and flightless I interacting protein 1), DDX41 (DEAD box polypeptide 41) (“DEAD” disclosed as SEQ ID NO: 51), and members of the HIN-200 (hematopoietic interferon-inducible nuclear proteins) family such as AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16). Molecular information is available on the HIN domain family as reflected in structures of their complexes with dsDNA. A requirement for multiple sensors may be a reflection of distinctive cell-type specific activities. Cytoplasmic detection of dsDNA activates stimulator of interferon genes (STING) in the cytoplasm, which in turn initiates a cascade of events by first activating kinases IKK (IκB kinase) and TBK1 (TANK-binding kinase 1), leading to phosphorylation and activation of the transcription factors NF-κB (nuclear factor κB) and IRF3 (interferon regulatory factor). These phosphorylated transcription factors translocate to the nucleus to target immune and inflammatory genes leading to the production of cytokines and type I interferons, thereby triggering the host immune response. Therefore, there is a need for therapeutic agents to modulate the induction of interferon and other relevant components in these pathways.